Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases have been identified thus far. Expansion of a CGG.CCG-repeat in the 5'UTR of the FMR1 gene is associated with 3 different clinical presentations: Individuals with 55-200 repeats, the so-called premutation allele, are at risk for Fragile X-associated tremor-ataxia syndrome whose symptoms include in addition to neurodegeneration, loss of autonomic function including bowel and urinary incontinence. Female carriers of premutation alleles are also at risk of FX-associated primary ovarian insufficiency. Furthermore, in females, the premutation allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is associated with intellectual disability, autistic symptoms, digestive difficulties, and behavior problems including aggression and depression. The mechanism by which is expansion occurs is unknown. It is thought to differ from the generalized microsatellite instability seen in many different cancers in that the instability is confined to a single genetic locus and shows a strong expansion bias. Repeat expansion diseases caused by small increases in repeat number frequently show a paternal bias, while those diseases resulting from large increases in repeat number are frequently maternally derived. Whether this reflects 2 different mechanisms is unknown. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. This site, which gives the disorder its name, is one of many fragile sites present on the human genome. These sites are prone to breakage and in some cases are associated with deleterious chromosome deletions and translocations. Progress report: We have generated FXS premutation mice containing 120 CGG.CCG-repeats in the 5 UTR of the endogenous murine Fmr1 gene (Entezam et al., 2007). We have shown that the repeat in these animals shows instability resembling the instability seen in human carriers of premutation alleles in its frequency and expansion bias. However most of these expansions are small and have a paternal expansion bias. This is reminiscent of what is seen in human carriers of smaller premutation alleles. In 2008 we showed that ATR, a key DNA damage checkpoint protein is involved in protecting the genome against a class of expansions that are exclusively maternally transmitted (Entezam and Usdin, 2008). In this reporting period we showed that a second DNA damage checkpoint protein, ATM is involved in blocking a second form of repeat expansion, one that occurs in both males and females but has a paternal expansion bias (Entezam and Usdin, 2009). The ATM-sensitive step is confined to the diploid gamete. When this step fails, ATM-independent repair is possible in the haploid gamete or early embryo. However, we showed that while this repair occurred quite efficiently in female embryos, it was less efficient in male embryos. This suggests that the process that gives rise to these expansions may be related to the activity of an X-linked DNA repair gene. Work is underway to test this hypothesis. We have also shown that agents that induce fragility of the Fragile X fragile site activate both the ATR and the ATM DNA damage response pathways (Kumari et. al., 2009). This suggests that similar mechanisms may be responsible for both chromosome fragility and repeat expansion. ATR acts to reduce fragility induced by fluorodeoxyuridine (FdU), while ATM exacerbates it. ATM also reduces a second, new, form of chromosome fragility that occurs spontaneously. Work is underway to try and understand the molecular basis of the 2 different fragile sites.